Ruth Seelige

Role of the Heparan Sulfate Proteoglycan Syndecan-1 (CD138) in Delayed-Type Hypersensitivity

The cell surface heparan sulfate proteoglycan syndecan-1 (CD138) modulates the activity of chemokines, cytokines, integrins, and other adhesion molecules which play important roles in the regulation of inflammation. We have previously shown that syndecan-1-deficient murine leukocytes display increased interactions with endothelial cells and increased diapedesis in vivo and in vitro. In this study, we demonstrate that syndecan-1 has an important function as a negative modulator in the murine contact allergy model of oxazolone-mediated delayed-type hypersensitivity (DTH). Following elicitation of the DTH response, syndecan-1-deficient mice showed an increase in leukocyte recruitment, resulting in an increased and prolonged edema formation. Expression of the cytokines TNF-α and IL-6 of the chemokines CCL5/RANTES and CCL-3/MIP-1α and of the adhesion molecule ICAM-1 were significantly increased in syndecan-1-deficient compared with wild-type mice. In wild-type mice, syndecan-1 mRNA and protein expression was reduced during the DTH response. The differentially increased adhesion of syndecan-1-deficient leukocytes to ICAM-1 was efficiently inhibited in vitro by CD18-blocking Abs, which emerges as one mechanistic explanation for the anti-inflammatory effects of syndecan-1. Collectively, our results show an important role of syndecan-1 in the contact DTH reaction, identifying syndecan-1 as a novel target in anti-inflammatory therapy.

Cutting Edge: Endothelial-Specific Gene Ablation of CD99L2 Impairs Leukocyte Extravasation In Vivo

CD99-like 2 (CD99L2) is a membrane protein with moderate sequence homology to CD99, which initiates cell aggregation of transfected cells and that is strongly expressed on endothelial cells, neutrophils, and lymphocytes. We showed recently that Abs against CD99L2 inhibit neutrophil, but not T lymphocyte, recruitment into inflamed tissues. In this study, we have generated conditional gene–deficient mice for CD99L2 and show by analyzing them in various inflammation models several results. First, gene ablation of CD99L2 impairs neutrophil recruitment into inflamed cremaster and peritoneum. Second, despite the strong expression of CD99L2 on peripheral neutrophils, only gene ablation on endothelial cells but not on myeloid cells affects neutrophil extravasation. Third, in contrast to our previous Ab-based results, recruitment of activated T cells into inflamed skin was impaired in mice lacking CD99L2 on endothelial cells. We conclude that CD99L2 is an essential endothelial Ag for leukocyte extravasation, which does not require homophilic interactions with CD99L2 on leukocytes.

Heparan sulfate expression in the neural crest is essential for mouse cardiogenesis

Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1(-/-) embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect.

Mechanisms regulating immune surveillance of cellular stress in cancer

The purpose of this review is to explore immune-mediated mechanisms of stress surveillance in cancer, with particular emphasis on the idea that all cancers have classical hallmarks (Hanahan and Weinberg in Cell 100:57–70, 67; Cell 144:646–674, 68) that could be interrelated. We postulate that hallmarks of cancer associated with cellular stress pathways (Luo et al. in Cell 136:823–837, 101) including oxidative stress, proteotoxic stress, mitotic stress, DNA damage, and metabolic stress could define and modulate the inflammatory component of cancer. As such, the overarching goal of this review is to define the types of cellular stress that cancer cells undergo, and then to explore mechanisms by which immune cells recognize, respond to, and are affected by each stress response.

The ancient cytokine IL-17D is regulated by Nrf2 and mediates tumor and virus surveillance

Early stage immune responses can dictate the severity and outcome of inflammatory processes such as tumor growth and viral infection. Cytokines such as the interleukin 17 (IL-17) family and cellular stress defense (e.g., anti-oxidant) pathways have evolved early and regulate disease surveillance in vertebrates and invertebrates as far back as Caenorhabditis elegans. Our group has recently found a new role for nuclear factor erythroid-derived 2-like 2 (Nrf2) in regulating early anti-cancer immune responses by inducing IL-17D and recruiting natural killer (NK) cells. In this Cytokine Stimulus, we discuss recent findings that encourage boosting the Nrf2/IL-17D/NK cell axis for the treatment of cancer and viral infection.

Endothelial CD99 supports arrest of mouse neutrophils in venules and binds to neutrophil PILRs

CD99 is a crucial regulator of the transmigration (diapedesis) of leukocytes through the blood vessel wall. Here, we report that CD99 acts at 2 different steps in the extravasation process. In agreement with previous antibody-blocking experiments, we found that CD99 gene inactivation caused neutrophil accumulation between venular endothelial cells and the basement membrane in the inflamed cremaster. Unexpectedly, we additionally found that leukocyte attachment to the luminal surface of the venular endothelium was impaired in the absence of CD99. Intravital video microscopy revealed that CD99 supported rapid chemokine-induced leukocyte arrest. Inhibition of leukocyte attachment and extravasation were both solely due to the absence of CD99 on endothelial cells, whereas CD99 on leukocytes was irrelevant. Therefore, we searched for heterophilic ligands of endothelial CD99 on neutrophils. We found that endothelial cells bind to the paired immunoglobulinlike receptors (PILRs) in a strictly CD99-dependent way. In addition, endothelial CD99 was coprecipitated with PILRs from neutrophils that adhered to endothelial cells. Furthermore, soluble CD99 carrying a transferable biotin tag could transfer this tag covalently to PILR when incubated with intact neutrophils. Binding of neutrophils under flow to a surface coated with P-selectin fragment crystallizable (Fc) and intercellular adhesion molecule 1 (ICAM-1) Fc became more shear resistant if CD99 Fc was coimmobilized. This increased shear resistance was lost if neutrophils were preincubated with anti-PILR antibodies. We concluded that endothelial CD99 promotes leukocyte attachment to endothelium in inflamed vessels by a heterophilic ligand. In addition, CD99 binds to PILRs on neutrophils, an interaction that leads to increased shear resistance of the neutrophil attachment to ICAM-1.

Interleukin-17D and Nrf2 mediate initial innate immune cell recruitment and restrict MCMV infection

Innate immune cells quickly infiltrate the site of pathogen entry and not only stave off infection but also initiate antigen presentation and promote adaptive immunity. The recruitment of innate leukocytes has been well studied in the context of extracellular bacterial and fungal infection but less during viral infections. We have recently shown that the understudied cytokine Interleukin (IL)-17D can mediate neutrophil, natural killer (NK) cell and monocyte infiltration in sterile inflammation and cancer. Herein, we show that early immune cell accumulation at the peritoneal site of infection by mouse cytomegalovirus (MCMV) is mediated by IL-17D. Mice deficient in IL-17D or the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an inducer of IL-17D, featured an early decreased number of innate immune cells at the point of viral entry and were more susceptible to MCMV infection. Interestingly, we were able to artificially induce innate leukocyte infiltration by applying the Nrf2 activator tert-butylhydroquinone (tBHQ), which rendered mice less susceptible to MCMV infection. Our results implicate the Nrf2/IL-17D axis as a sensor of viral infection and suggest therapeutic benefit in boosting this pathway to promote innate antiviral responses.

CD99L2 deficiency inhibits leukocyte entry into the central nervous system and ameliorates neuroinflammation

Leukocyte entry into the CNS is a crucial step in the development of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Adhesion molecules mediating the docking of leukocytes to the endothelium of the blood–brain barrier (BBB) represent valuable targets for interference with the disease. However, little is known about the adhesion and signaling mechanisms in endothelial cells that mediate the diapedesis through the BBB. Here, we show that conditional Tie‐2‐Cre driven gene inactivation of CD99L2 inhibits leukocyte entry into the CNS during active MOG35‐55‐induced EAE and alleviates severity of the disease. No detrimental effect on the immune response was observed. The number of perivascular cuffs around vessels of the CNS was reduced, as was the number of inflammatory foci, sites of demyelination and expression levels of pro‐inflammatory cytokines. Three‐dimensional analysis of vibratome sections of the CNS revealed an accumulation of leukocytes between endothelial cells and the underlying basement membrane, whereas leukocyte docking to the luminal surface of the endothelium of the BBB was unaffected. Collectively, these results suggest that CD99L2 participates in the development of EAE by supporting diapedesis of leukocytes through the endothelial basement membrane of blood vessels of the BBB in the CNS.

Abstract 4698: Immuno-oncological efficacy of RXDX-106, a novel, selective and potent small molecule TAM (TYRO3, AXL, MER) inhibitor

The TAM family of receptor tyrosine kinases (RTKs), TYRO3, AXL, and MER, has been implicated in the pathogenesis and progression of many cancer types. In cancer cells, overexpression of TAM RTKs is associated with resistance and mesenchymal phenotype. In immune cells, TAM RTKs play a key homeostatic role as negative regulators of immune responses, contributing to the evasion of cancer cells from immune surveillance. Here we studied expression as well as functional modulation of TAM RTKs on immune cells in the tumor microenvironment and evaluated whether RXDX-106, a selective and potent, pseudo-irreversible small molecule inhibitor of TAM RTKs could restore and enhance host immunity against cancer. We first evaluated the expression of TAM RTKs on immune cells in non-tumor bearing mice and tumor bearing mice. Tumor bearing animals showed increased expression of TAM RTKs in subsets of immune cells such as lymph node macrophages and T cells, compared to non-tumor bearing mice. Particularly, TAM expression was upregulated in CD8+ T cells in tumors, suggesting that the increased expression could restrain T cell activation and mediate exhaustion. RXDX-106 reduced TAM phosphorylation on macrophages at concentrations as low as 2.5 nM, and inhibited AXL- and MER-dependent in vitro phagocytosis with an IC 50 of 10.8 and 9.1 nM, respectively. In syngeneic mouse models, including 4T-1 breast cancer model and CT26 colon cancer model, RXDX-106 dose-dependently inhibited tumor growth and demonstrated further tumor growth inhibition in combination with anti-CTLA-4 or anti-PD-1 antibodies. The tumor growth inhibition was associated with increased total T cells, decreased T-regulatory cells, and increased IFN-γ production by NK cells in the tumor. RXDX-106 also increased IFN-γ levels in the blood when combined with anti-PD-1 antibody. Although RXDX-106 had no effect on T cell proliferation in vitro, it could increase the proliferation of T cells in lymph nodes in vivo. In conclusion, we demonstrated that tumors could induce TAM expression on subsets of T cells and that inhibition of TAM RTK activity by the small molecule inhibitor, RXDX-106, could potentially remove the molecular “brake” on immune activation in macrophages, NK cells and T cells, resulting in repolarization of the immune response towards an anti-tumor environment. The unique mechanism of activating both innate and acquired immunity by RXDX-106, and the compelling preclinical data in cell line and syngeneic models, support the clinical development of RXDX-106 in a wide variety of cancers. Citation Format: Yumi Yokoyama, Erin D. Lew, Ruth Seelige, Colin Walsh, Maria Barrera, Elizabeth Tindall, Joanne Oh, Heather Ely, Amy Diliberto, Amanda Albert, Jack Bui, Gary Li. Immuno-oncological efficacy of RXDX-106, a novel, selective and potent small molecule TAM (TYRO3, AXL, MER) inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4698. doi:10.1158/1538-7445.AM2017-4698